Polyethylene glycol and caspase inhibitor emricasan alleviate cold injury in primary rat hepatocytes.

Current methods of storing explanted donor livers at 4 °C in University of Wisconsin (UW) solution result in loss of graft function and ultimately lead to less-than-ideal outcomes post transplantation. Our lab has previously shown that supplementing UW solution with 35-kilodalton polyethylene glycol (PEG) has membrane stabilizing effects for cold stored primary rat hepatocytes in suspension. Expanding on past studies, we here investigate if PEG has the same beneficial effects in an adherent primary rat hepatocyte cold storage model. In addition, we investigated the extent of cold-induced apoptosis through treating cold-stored hepatocytes with pan caspase inhibitor emricasan. In parallel to storage at the current cold storage standard of 4 °C, we investigated the effects of lowering the storage temperature to -4 °C, at which the storage solution remains ice-free due to the supercooling phenomenon. We show the addition of 5 % PEG to the storage medium significantly reduced the release of lactate dehydrogenase (LDH) in plated rat hepatocytes and a combinatorial treatment with emricasan maintains hepatocyte viability and morphology following recovery from cold storage. These results show that cold-stored hepatocytes undergo multiple mechanisms of cold-induced injury and that PEG and emricasan treatment in combination with supercooling may improve cell and organ preservation.

Role of PEG35, Mitochondrial ALDH2, and Glutathione in Cold Fatty Liver Graft Preservation: An IGL-2 Approach.

The total damage inflicted on the liver before transplantation is associated with several surgical manipulations, such as organ recovery, washout of the graft, cold conservation in organ preservation solutions (UW, Celsior, HTK, IGL-1), and rinsing of the organ before implantation. Polyethylene glycol 35 (PEG35) is the oncotic agent present in the IGL-1 solution, which is an alternative to UW and Celsior solutions in liver clinical transplantation. In a model of cold preservation in rats (4 °C; 24 h), we evaluated the effects induced by PEG35 on detoxifying enzymes and nitric oxide, comparing IGL-1 to IGL-0 (which is the same as IGL-1 without PEG). The benefits were also assessed in a new IGL-2 solution characterized by increased concentrations of PEG35 (from 1 g/L to 5 g/L) and glutathione (from 3 mmol/L to 9 mmol/L) compared to IGL-1. We demonstrated that PEG35 promoted the mitochondrial enzyme ALDH2, and in combination with glutathione, prevented the formation of toxic aldehyde adducts (measured as 4-hydroxynonenal) and oxidized proteins (AOPP). In addition, PEG35 promoted the vasodilator factor nitric oxide, which may improve the microcirculatory disturbances in steatotic grafts during preservation and revascularization. All of these results lead to a reduction in damage inflicted on the fatty liver graft during the cold storage preservation. In this communication, we report on the benefits of IGL-2 in hypothermic static preservation, which has already been proved to confer benefits in hypothermic oxygenated dynamic preservation. Hence, the data reported here reinforce the fact that IGL-2 is a suitable alternative to be used as a unique solution/perfusate when hypothermic static and preservation strategies are used, either separately or combined, easing the logistics and avoiding the mixture of different solutions/perfusates, especially when fatty liver grafts are used. Further research regarding new therapeutic and pharmacological insights is needed to explore the underlying mitochondrial mechanisms exerted by PEG35 in static and dynamic graft preservation strategies for clinical liver transplantation purposes.

Porcine heart valve, aorta and trachea cryopreservation and thawing using polydimethylsiloxane.

The most common cryopreservation protocols of biological tissues suitable for their further implantation has some disadvantages and limited to one sample per procedure with no possible repeated freezing in case of clinical needs. This study is aimed to improve a biological tissues cryopreservation by adding a new heat transfer fluid - polydimethylsiloxane (PDMS). To evaluate its efficiency the porcine biological tissues (heart valves, aortic and trachea fragments) were cryopreserved and thawed in low-viscous PDMS. According to the computer simulation, the midsection cooling rate was up to 490 °C/min and the midsection thawing rate was up to 1140 °C/min with admissible temperature uniformity. Cryoprotectants and liquid nitrogen were not used. The quality of tissue cryopreservation was evaluated using a number of histological and immunohistochemical methods (Orcein, H&E, Anti-CD34, Anti-Vimentin, Anti-Actin staining). Cryopreserved tissues showed no significant morphological difference in comparison with control group both in case of immediate thawing, and after 2 months of low temperature storage. Computer simulation of heat transfer showed the thermal limitations of used approach for larger specimens. The use of PDMS is proposed for preservation of vascular tissue in order to implant it in the form of homotransplants or biobanking with the possible additional use of an internal hydrophilic coating to prevent hydrophobization.

Polyethylene Glycol 35 as a Perfusate Additive for Mitochondrial and Glycocalyx Protection in HOPE Liver Preservation.

Organ transplantation is a multifactorial process in which proper graft preservation is a mandatory step for the success of the transplantation. Hypothermic preservation of abdominal organs is mostly based on the use of several commercial solutions, including UW, Celsior, HTK and IGL-1. The presence of the oncotic agents HES (in UW) and PEG35 (in IGL-1) characterize both solution compositions, while HTK and Celsior do not contain any type of oncotic agent. Polyethylene glycols (PEGs) are non-immunogenic, non-toxic and water-soluble polymers, which present a combination of properties of particular interest in the clinical context of ischemia-reperfusion injury (IRI): they limit edema and nitric oxide induction and modulate immunogenicity. Besides static cold storage (SCS), there are other strategies to preserve the organ, such as the use of machine perfusion (MP) in dynamic preservation strategies, which increase graft function and survival as compared to the conventional static hypothermic preservation. Here we report some considerations about using PEG35 as a component of perfusates for MP strategies (such as hypothermic oxygenated perfusion, HOPE) and its benefits for liver graft preservation. Improved liver preservation is closely related to mitochondria integrity, making this organelle a good target to increase graft viability, especially in marginal organs (e.g., steatotic livers). The final goal is to increase the pool of suitable organs, and thereby shorten patient waiting lists, a crucial problem in liver transplantation.

The Novel N,N-bis-2-Hydroxyethyl-2-Aminoethanesulfonic Acid-Gluconate-Polyethylene Glycol-Hypothermic Machine Perfusion Solution Improves Static Cold Storage and Reduces Ischemia/Reperfusion Injury in Rat Liver Transplant.

Organ transplantation is the treatment of choice against terminal and irreversible organ failure. Optimal preservation of the graft is crucial to counteract cold ischemia effects. As we developed an N,N-bis-2-hydroxyethyl-2-aminoethanesulfonic acid-gluconate-polyethylene glycol (BGP)-based solution (hypothermic machine perfusion [HMP]), we aimed to analyze the use of this solution on static cold storage (SCS) of rat livers for transplantation as compared with the histidine tryptophan ketoglutarate (HTK) preservation solution. Livers procured from adult male Sprague Dawley rats were preserved with BGP-HMP or HTK solutions. Liver total water content and metabolites were measured during the SCS at 0°C for 24 hours. The function and viability of the preserved rat livers were first assessed ex vivo after rewarming (90 minutes at 37°C) and in vivo using the experimental model of reduced-size heterotopic liver transplantation. After SCS, the water and glycogen content in both groups remained unchanged as well as the tissue glutathione concentration. In the ex vivo studies, livers preserved with the BGP-HMP solution were hemodynamically more efficient and the O2 consumption rate was higher than in livers from the HTK group. Bile production and glycogen content after 90 minutes of normothermic reperfusion was diminished in both groups compared with the control group. Cellular integrity of the BGP-HMP group was better, and the histological damage was reversible. In the in vivo model, HTK-preserved livers showed a greater degree of histological injury and higher apoptosis compared with the BGP-HMP group. In conclusion, our results suggest a better role of the BGP-HMP solution compared with HTK in preventing ischemia/reperfusion injury in the rat liver model.

Normothermic machine perfusion of donor livers without the need for human blood products.

Normothermic machine perfusion (NMP) enables viability assessment of donor livers prior to transplantation. NMP is frequently performed by using human blood products including red blood cells (RBCs) and fresh frozen plasma (FFP). Our aim was to examine the efficacy of a novel machine perfusion solution based on polymerized bovine hemoglobin-based oxygen carrier (HBOC)-201. Twenty-four livers declined for transplantation were transported by using static cold storage. Upon arrival, livers underwent NMP for 6 hours using pressure-controlled portal and arterial perfusion. A total of 12 livers were perfused using a solution based on RBCs and FFPs (historical cohort), 6 livers with HBOC-201 and FFPs, and another 6 livers with HBOC-201 and gelofusine, a gelatin-based colloid solution. Compared with RBC + FFP perfused livers, livers perfused with HBOC-201 had significantly higher hepatic adenosine triphosphate content, cumulative bile production, and portal and arterial flows. Biliary secretion of bicarbonate, bilirubin, bile salts, and phospholipids was similar in all 3 groups. The alanine aminotransferase concentration in perfusate was lower in the HBOC-201-perfused groups. In conclusion, NMP of human donor livers can be performed effectively using HBOC-201 and gelofusine, eliminating the need for human blood products. Perfusing livers with HBOC-201 is at least similar to perfusion with RBCs and FFP. Some of the biomarkers of liver function and injury even suggest a possible superiority of an HBOC-201-based perfusion solution and opens a perspective for further optimization of machine perfusion techniques. Liver Transplantation 24 528-538 2018 AASLD.

The Optimal PEG for Kidney Preservation: A Preclinical Porcine Study.

University of Wisconsin (UW) solution is not optimal for preservation of marginal organs. Polyethylene glycol (PEG) could improve protection. Similarly formulated solutions containing either 15 or 20 g/L PEG 20 kDa or 5, 15 and 30 g/L PEG 35 kDa were tested in vitro on kidney endothelial cells, ex vivo on preserved kidneys, and in vivo in a pig kidney autograft model. In vitro, all PEGs provided superior preservation than UW in terms of cell survival, adenosine triphosphate (ATP) production, and activation of survival pathways. Ex vivo, tissue injury was lower with PEG 20 kDa compared to UW or PEG 35 kDa. In vivo, function recovery was identical between UW and PEG 35 kDa groups, while PEG 20 kDa displayed swifter recovery. At three months, PEG 35 kDa 15 and 30 g/L animals had worse outcomes than UW, while 5 g/L PEG 35 kDa was similar. PEG 20 kDa was superior to both UW and PEG 35 kDa in terms of function and fibrosis development, with low activation of damage pathways. PEG 20 kDa at 15 g/L was superior to 20 g/L. While in vitro models did not discriminate between PEGs, in large animal models of transplantation we showed that PEG 20 kDa offers a higher level of protection than UW and that longer chains such as PEG 35 kDa must be used at low doses, such as found in Institut George Lopez (IGL1, 1g/L).

Normothermic ex vivo liver perfusion using steen solution as perfusate for human liver transplantation: First North American results.

The European trial investigating normothermic ex vivo liver perfusion (NEVLP) as a preservation technique for liver transplantation (LT) uses gelofusine, a non-US Food and Drug Administration-approved, bovine-derived, gelatin-based perfusion solution. We report a safety and feasibility clinical NEVLP trial with human albumin-based Steen solution. Transplant outcomes of 10 human liver grafts that were perfused on the Metra device at 37 °C with Steen solution, plus 3 units of erythrocytes were compared with a matched historical control group of 30 grafts using cold storage (CS) as the preservation technique. Ten liver grafts were perfused for 480 minutes (340-580 minutes). All livers cleared lactate (final lactate 1.46 mmol/L; 0.56-1.74 mmol/L) and produced bile (61 mL; 14-146 mL) during perfusion. No technical problems occurred during perfusion, and all NEVLP-preserved grafts functioned well after LT. NEVLP versus CS had lower aspartate aminotransferase and alanine aminotransferase values on postoperative days 1-3 without reaching significance. No difference in postoperative graft function between NEVLP and CS grafts was detected as measured by day 7 international normalized ratio (1.1 [1-1.56] versus 1.1 [1-1.3]; P = 0.5) and bilirubin (1.5; 1-7.7 mg/dL versus 2.78; 0.4-15 mg/dL; P = 0.5). No difference was found in the duration of intensive care unit stay (median, 1 versus 2 days; range, 0-8 versus 0-23 days; P = 0.5) and posttransplant hospital stay (median, 11 versus 13 days; range, 8-17 versus 7-89 days; P = 0.23). Major complications (Dindo-Clavien ≥ 3b) occurred in 1 patient in the NEVLP group (10%) compared with 7 (23%) patients in the CS group (P = 0.5). No graft loss or patient death was observed in either group. Liver preservation with normothermic ex vivo perfusion with the Metra device using Steen solution is safe and results in comparable outcomes to CS after LT. Using US Food and Drug Administration-approved Steen solution will avoid a potential regulatory barrier in North America. Liver Transplantation 22 1501-1508 2016 AASLD.

Polyethylene glycol protects primary hepatocytes during supercooling preservation.

Cold storage (at 4°C) offers a compromise between the benefits and disadvantages of cooling. It allows storage of organs or cells for later use that would otherwise quickly succumb to warm ischemia, but comprises cold ischemia that, when not controlled properly, can result in severe damage as well by both similar and unique mechanisms. We hypothesized that polyethylene glycol (PEG) 35 kDa would ameliorate these injury pathways and improve cold primary hepatocyte preservation. We show that reduction of the storage temperature to below zero by means of supercooling, or subzero non-freezing, together with PEG supplementation increases the viable storage time of primary rat hepatocytes in University of Wisconsin (UW) solution from 1 day to 4 days. We find that the addition of 5% PEG 35 kDa to the storage medium prevents cold-induced lipid peroxidation and maintains hepatocyte viability and functionality during storage. These results suggest that PEG supplementation in combination with supercooling may enable a more optimized cell and organ preservation.

Dehydration of corneal anterior donor tissue with polyethylene glycol (PEG)-enriched media.

Anterior donor grafts (including scleral rim, without Descemet membrane) increase in thickness and become hazy upon storage in organ culture (OC) medium. Transfer of these grafts to standard dehydration media just before transplantation does not reduce their thickness to normal. Therefore, we assessed the efficacy of different media enriched with polyethylene glycol (PEG) as dehydrating agents for organ-cultured anterior donor grafts. Grafts were harvested and stored in the commercial OC medium 'Max' (without dextran) for 1 week, and subsequently dehydrated in the standard commercial dehydration medium 'Jet' (with dextran) supplemented with 4-20% PEG3350, or 'Max' supplemented with 20% PEG6000 and PEG20.000, or 5-20% PEG35.000. Central corneal thickness (CCT), as assessed by anterior segment-optical coherence tomography, and transparency were evaluated before, and at 1, 4 and 7 days of dehydration. Transfer of grafts after 1 week of OC (average 1,200 µm) to 'Jet' supplemented with PEG3350 revealed a concentration-dependent effect of dehydration; CCT was restored to normal (500-600 µm) when 10% PEG3350 was added. However, transparency was only temporarily restored; after 1 day, the grafts turned hazy. In contrast, grafts transferred to 'Max' supplemented with 20% PEG35.000 were transparent throughout the evaluation period, but were dehydrated to beyond normal levels (average 300 µm). 'Max' supplemented with 5% PEG35.000 dehydrated grafts to normal values and restored transparency throughout. Thus, dehydration of anterior donor grafts prior to surgery in dextran-free OC medium supplemented with 5% PEG35.000 reduces graft thickness to normal and may facilitate anterior keratoplasty procedures.

Comparative study of indigenously prepared and imported, demineralized, freeze-dried, irradiated bone allograft in the treatment of periodontal infrabony defects.

Demineralized freeze-dried bone allograft (DFDBA) has been used extensively in periodontal therapy. Questions have been raised however, about the osteogenic potential of the variety of grafts available. In India the cost factor is another important consideration. The aim of this study therefore was to evaluate the clinical efficiency of the low priced, indigenously prepared DFDBA obtained from the Tata Memorial Hospital (TMH) Tissue Bank, in periodontal regeneration in infrabony periodontal defects, as compared to DFDBA obtained from the Pacific Coast Tissue Bank (DEMBONE). The latter was used as the control. 16 patients with bilaterally similar periodontal infrabony defects were selected, and randomly allotted to the test and control groups. At baseline, using standardized protocol, recession, probing depths (PD), and clinical attachment levels (CAL) were measured, following which periodontal surgery was carried out, with placement of the respective graft materials. Patients were recalled after 6 months for re-assessment. Statistically significant improvement was obtained for PD reduction and CAL gain for both groups (p < 0.05). However, no significant difference was observed between the test and control groups. It was therefore concluded that both the materials from different tissue banks are equally effective clinically, with the test material being additionally cost effective.

The Effects of Oxygen-Derived Free-Radical Scavengers During Normothermic Ex-Situ Heart Perfusion.

Oxidative stress occurs during ex-situ heart perfusion (ESHP) and may negatively affect functional preservation of the heart. We sought to assess the status of key antioxidant enzymes during ESHP, and the effects of augmenting these antioxidants on the attenuation of oxidative stress and improvement of myocardial and endothelial preservation in ESHP. Porcine hearts were perfused for 6 hours with oxygen-derived free-radical scavengers polyethylene glycol (PEG)-catalase or PEG-superoxide dismutase (SOD) or with naive perfusate (control). The oxidative stress-related modifications were determined in the myocardium and coronary vasculature, and contractile function, injury, and endothelial integrity were compared between the groups. The activity of key antioxidant enzymes decreased and adding catalase and SOD restored the enzyme activity. Cardiac function and endothelial integrity were preserved better with restored catalase activity. Catalase and SOD both decreased myocardial injury and catalase reduced ROS production and oxidative modification of proteins in the myocardium and coronary vasculature. The activity of antioxidant enzymes decrease in ESHP. Catalase may improve the preservation of cardiac function and endothelial integrity during ESHP. While catalase and SOD may both exert cardioprotective effects, unbalanced SOD and catalase activity may paradoxically increase the production of reactive species during ESHP.

High molecular weight polyethylene glycol (PEG 15-20) maintains mucosal microbial barrier function during intestinal graft preservation.

BACKGROUND: During organ transplantation, it is inevitable that tissues undergo cold ischemia during harvest and transport before implantation. Polyethylene-based polymers have been proposed and tested as preservation agents, with promising results. We have previously reported that a high molecular weight polyethylene glycol (PEG) (15-20,000 MW; PEG 15-20) protects the intestinal epithelium against a variety of cellular stresses, including radiation injury and microbial invasion, by mechanisms that appear to involve lipid rafts. The aim of this study was to determine the preservation effect of PEG 15-20 on the integrity of intestine grafts harvested for subsequent transplantation. MATERIALS AND METHODS: We harvested intestinal grafts from mice using a complete surgical technique for intestinal transplantation and assessed them for the effect of PEG on graft tissue integrity. We preserved half of the grafts in histidine-tryptophan-ketoglutarate solution (HTK) alone and half in HTK-PEG 15-20 solution at 4°C for 24 h. We examined gross morphology, wet to dry ratios, histology, terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine, 5'-triphosphate nick end labeling assay for apoptosis, goblet cell numbers, and bacterial localization studies to evaluate the effect of PEG on tissue integrity. RESULTS: Results demonstrated that PEG 15-20 had a superior preservation effect over HTK alone in all parameters tested. The effect of PEG was notable on attenuation of epithelial apoptosis, preservation of mucus-producing cells, and bacterial adherence to the epithelium. CONCLUSIONS: Taken together, these studies suggest that use of PEG 15-20 as a potential adjuvant during intestinal transplant may offer significant promise to prolong graft survival during organ harvest.

Iliac crest fresh-frozen allografts and autografts in maxillary and mandibular reconstruction: a histologic and histomorphometric evaluation.

AIM: The aim of this paper was to compare histologically and histomorphometrically the osseointegration of iliac crest fresh-frozen allografts and autografts in human pre-prosthetic maxillary and mandibular onlay bone blocks reconstruction. METHODS: Twelve patients with edentulous atrophic ridges, scheduled for implant-supported prosthetic restorations, underwent reconstruction using iliac crest fresh-frozen allografts (group A, six patients) or autografts (group B, six patients). Four-to-nine months later implants were placed in the augmented areas and bone specimens were simultaneously obtained using trephine burs. The specimens were processed for ground sections and evaluated histologically and histomorphometrically. RESULTS: The postoperative course was uneventful in all patients in group B and in all except one in group A. Late complications occurred in 5 patients of group A. Dental implants could be inserted in all cases. Specimens from group A showed a vascularized bone with osteoprogenitor stem cells and medium-high grade of bone remodeling. Small areas of necrotic bone were observed sporadically. Sections obtained from group B revealed an advanced stage of bone remodeling. The histomorphometric analysis showed in group A a mean proportion of 24.7±14.7% for lamellar bone, 28.4±13.3% for newly formed bone and 46.9±16.9% for bone marrow; in group B the corresponding values were 25.3±15.3%, 22.9±11.0%, 51.7±15.7%. No statistically significant difference was found (Wilcoxon Test; P>0.05). CONCLUSION: There were no significant histological differences between group A and B. Larger studies with long term follow-up are needed to confirm that fresh-frozen allografts are a reliable alternative to autografts.

Normothermic Ex Vivo Liver Machine Perfusion in Mouse.

This protocol presents an optimized erythrocytes-free NEVLP system using mouse livers. Ex vivo preservation of mouse livers was achieved by employing modified cannulas and techniques adapted from conventional commercial ex vivo perfusion equipment. The system was utilized to evaluate the preservation outcomes following 12 h of perfusion. C57BL/6J mice served as liver donors, and the livers were explanted by cannulating the portal vein (PV) and bile duct (BD), and subsequently flushing the organ with warm (37 °C) heparinized saline. Then, the explanted livers were transferred to the perfusion chamber and subjected to normothermic oxygenated machine perfusion (NEVLP). Inlet and outlet perfusate samples were collected at 3 h intervals for perfusate analysis. Upon completion of the perfusion, liver samples were obtained for histological analysis, with morphological integrity assessed using modified Suzuki-Score through Hematoxylin-Eosin (HE) staining. The optimization experiments yielded the following findings: (1) mice weighing over 30 g were deemed more suitable for the experiment due to the larger size of their bile duct (BD). (2) a 2 Fr (outer diameter = 0.66 mm) polyurethane cannula was better suited for cannulating the portal vein (PV) when compared to a polypropylene cannula. This was attributed to the polyurethane material's enhanced grip, resulting in reduced catheter slippage during the transfer from the body to the organ chamber. (3) for cannulation of the bile duct (BD), a 1 Fr (outer diameter = 0.33 mm) polyurethane cannula was found to be more effective compared to the polypropylene UT - 03 (outer diameter = 0.30 mm) cannula. With this optimized protocol, mouse livers were successfully preserved for a duration of 12 h without significant impact on the histological structure. Hematoxylin-Eosin (HE) staining revealed a well-preserved morphological architecture of the liver, characterized by predominantly viable hepatocytes with clearly visible nuclei and mild dilation of hepatic sinusoids.

The international normalised ratio to monitor coagulation factor production during normothermic machine perfusion of human donor livers.

BACKGROUND: Normothermic machine perfusion (NMP) of donor livers allows for new diagnostic and therapeutic strategies. As the liver produces most of the haemostatic proteins, coagulation assays such as the International Normalised Ratio (INR) performed in perfusate may be useful to assess hepatocellular function of donor livers undergoing NMP. However, high concentrations of heparin and low levels of fibrinogen may affect coagulation assays. METHODS: Thirty donor livers that underwent NMP were retrospectively included in this study, of which 18 were subsequently transplanted. We measured INRs in perfusate in presence or absence of exogenously added fibrinogen and/or polybrene. Additionally, we prospectively included 14 donor livers that underwent NMP (of which 11 were transplanted) and measured INR using both a laboratory coagulation analyser and a point-of-care device. RESULTS: In untreated perfusate samples, the INR was above the detection limit in all donor livers. Addition of both fibrinogen and polybrene was required for adequate INR assessment. INRs decreased over time and detectable perfusate INR values were found in 17/18 donor livers at the end of NMP. INR results were similar between the coagulation analyser and the point-of-care device, but did not correlate with established hepatocellular viability criteria. CONCLUSIONS: Most of the donor livers that were transplanted showed a detectable perfusate INR at the end of NMP, but samples require processing to allow for INR measurements using laboratory coagulation analysers. Point-of-care devices bypass this need for processing. The INR does not correlate with established viability criteria and might therefore have additional predictive value.

Development and Characterization of a Nonelectronic Versatile Oxygenating Perfusion System for Tissue Preservation.

Oxygenated machine perfusion of human organs has been shown to improve both preservation quality and time duration when compared to the current gold standard: static cold storage. However, existing machine perfusion devices designed for preservation and transportation of transplantable organs are too complicated and organ-specific to merit use as a solution for all organs. This work presents a novel, portable, and nonelectronic device potentially capable of delivering oxygenated machine perfusion to a variety of organs. An innovative pneumatic circuit system regulates a compressed oxygen source that cyclically inflates and deflates silicone tubes, which function as both the oxygenator and perfusion pump. Different combinations of silicone tubes in single or parallel configurations, with lengths ranging from 1.5 to 15.2 m, were evaluated at varying oxygen pressures from 27.6 to 110.3 kPa. The silicone tubes in parallel configurations produced higher peak perfusion pressures (70% increase), mean flow rates (102% increase), and oxygenation rates (268% increase) than the single silicone tubes that had equivalent total lengths. While pumping against a vascular resistance element that mimicked a kidney, the device achieved perfusion pressures (8.4-131.6 mmHg), flow rates (2.0-40.2 mL min-1), and oxygenation rates (up to 388 µmol min-1) that are consistent with values used in previous kidney preservation studies. The nonelectronic device achieved those perfusion parameters using 4.4 L min-1 of oxygen to operate. These results demonstrate that oxygenated machine perfusion can be successfully achieved without any electronic components.

Hypothermic preservation of rat liver microorgans (LMOs) in bes-gluconate solution. Protective effects of polyethyleneglycol (PEG) on total water content and functional viability.

We have reported of an alternative solution to preserve hepatocytes that have three key components: gluconate, sucrose and an aminosulfonic acid (BGS solution). In order to extend the use of this solution to organs as the liver, we evaluate the effect of the addition of PEG of 8, 20 and 35 kDa to BG Solution on the total water content and functional viability of rat liver microorgans (LMOs). LMOs were preserved (48 h 0 ºC) in the following solutions: ViaSpan(®); BGS; BG plus 4% PEG 8000 (BG8); BG plus 4% PEG 20.000 (BG20) and BG plus 4% PEG 35.000 (BG35). LDH Release and Total Water Content showed a marked increase in LMOs preserved in BGS. This indicates that, in the absence of PEG, the tissue showed important cell membrane integrity deterioration and was incapable of regulating cell volume. After the preservation period, all groups were reoxygenated (120 min, 37 ºC, KHR) and Total Water Content, Glycogen Content and Oxygen Consumption were determined. After 120 min LMOs preserved in BG35 showed values of Oxygen Consumption similar to controls. On the other hand, LMOs preserved in BG8, BG20 and ViaSpan(®) showed oxygen consumption rates and glycogen content significantly smaller than controls. In conclusion, BG35 was the most effective preservation solution to protect LMOs against cold preservation injury due to ischemia and reoxygenation. It is a good alternative to ViaSpan(®) because of its higher buffer capacity, its best indexes of respiration activity and for being considerably less expensive.

In-vitro and in-vivo study of periodontal ligament cryopreserved with a magnetic field.

INTRODUCTION: The purpose of this study was to examine the effect of a new cryopreservation method with a magnetic field on periodontal regeneration in vitro and in vivo. METHODS: Human periodontal ligament cells were frozen in 10% dimethyl sulfoxide by using a programmed freezer with a magnetic field. Cells were cryopreserved for 3 days at -150°C. Immediately after thawing, collagen type I and alkaline phosphatase gene expression were determined by real-time polymerase chain reaction. Incisors were extracted from 15-week-old Wistar rats and cryopreserved or dried for 3 days. Then the incisors were replanted into the same sockets. Ninety days after transplantation, they were observed under light microscopy. RESULTS: There was no difference in the messenger RNA expression of collagen type I between the cryopreserved and the control groups. The expression of alkaline phosphatase messenger RNA in the cryopreserved group was slightly decreased compared with the control group. There was no progressive root resorption in the teeth that were replanted immediately (control group) or cryopreserved. However, there was widespread root resorption and ankylosis in the dried teeth. CONCLUSIONS: These results show that a magnetic field programmed freezer can be successfully used for cryopreservation of teeth.

Polyethylene glycol modified-albumin enhances the cold preservation properties of University of Wisconsin solution in rat liver and a hepatocyte cell line.

Liver grafts preserved in cold storage undergo changes mainly manifested by morphological modifications of the sinusoidal endothelium that result in poor graft function upon reperfusion. The present studies aimed to determine if the addition of polyethylene glycol-albumin to University of Wisconsin (Peg-AlbUW) solution ameliorates the cold preservation injuries of liver grafts. Rat livers were preserved cold with various preservation solutions and evaluated for weight changes and endothelial morphology. Solutions that preserved graft weight and endothelial morphology were tested in the isolated perfused rat liver model to assess graft function. A rat hepatocyte cell line was evaluated for both viability and glutathione concentrations emulating cold preservation and reperfusion conditions. Liver grafts preserved with Peg-AlbUW maintained their initial weight and showed a conserved endothelial morphology compared with liver grafts preserved in UW for 30 h (P<0.05). Liver grafts preserved with Peg-AlbUW had improved portal blood flow and bile secretion compared with liver grafts preserved in UW for 30 h (P<0.05). In vitro we noted comparable hepatocyte viability when cells were preserved in Peg-AlbUW versus UW under similar preservation conditions (P>0.05); glutathione concentrations (reduced and total) were significantly increased in hepatocytes preserved in 3% Peg-AlbUW compared with other preservation solutions (P<0.05). The addition of Peg-Alb to UW preservation solution ameliorated the cold preservation injuries of rat liver grafts as shown by stable liver graft weight, a better preservation of the endothelial morphology, improved portal vein blood flow, and increased bile secretion. Peg-Alb-UW solution improved the integrity of the glutathione redox buffer system of a hepatocyte cell line after cold storage and reperfusion.